Akane is an apple cultivar which has a characteristic that lateral flowers are naturally abscised after the central flower is pollinated. However, the lateral flowers of many apple cultivars, for example Fuji cultivar, are not abscised naturally, and they are pollinated and ripe to mature fruits. So, in an orchard the lateral fruits are abscised by treatment of abscission-inducing chemical, such as sevin. I performed suppression subtractive hybridization (SSH) and dot blotting to investigate the molecular mechanism of natural abscission in cv. Akane and compare the natural abscission with chemical-inducing one in cv. Fuji. From the dot blotting, some up- or down-regulated clones were partially sequenced and homologies were searched from GenBank. In Akane lateral flowers, putative ser/thr kinase (ASK), phosphate transporter (APT), chitinase, cellulase, ribonuclease PR-10 like protein, and unknown genes were up-regulated, and cytochrome P-450 monooxygenase, hemolysin, sorbitol dehydrogenase, glycerol kinase (AGK), and unknown genes were down-regulated. In the sevin-treated Fuji flowers, putative senescence-associated protein, ribosomal protein L19, chloroplast ribosomal protein, and unknown genes were up-regulated, and elongation factor 1-α, fructose 1,6-bisphosphate aldolase, RING zinc finger protein, and unknown genes were down-regulated. From the genes searched in Akane lateral flowers and sevin-treated Fuji flowers, natural abscission and chemical-inducing one seemed to occur in different mechanism since they didn`t share any up- or down-regulated genes each other. Cellulase, chitinase and ribonuclease PR-10 were known as related in cell wall loosening and defense process at the abscission zone. Sevin treatment seemed to influence the genes related in cell survival. The expression patterns of ASK, APT, and AGK were characterized by RT-PCR. ASK and APT were up-regulated, and AGK was down-regulated specifically in Akane lateral flowers. Each gene was cloned from cDNA library and genomic DNA library. AGK and APT have high homologies with other glycerol kinases or phosphate transporters. However, ASK has low homology in C-terminal region compared to genes from other species and its function is not yet known. Cloned genes were used to construct binary vectors to transform micro-tomato and observe abscission-related or other distinct phenotypes in transformants. Two ASK transformants were obtained, which preliminarily showed different phenotypes compared to the wild type than wild type. One showed a little bit smaller in size than wild type, and the other had much more leaves. In the process of acclimation, 2 flowers were naturally abscised after flowering. Also, flowering and bearing fruits were poor. Three APT transformants were obtained, however they didn`t show any significant phenotype yet.
사과 아카네 품종은 후지와 같은 품종들과 달리 중심화가 수정된 후 열매로 성숙하면서 측화에서 자연 탈리 현상이 나타난다. 과수원에서는 측과로의 양분의 분산을 막기 위해 sevin 과 같은 적과제를 처리해주거나 수작업으로 측화를 따주어 중심화만을 남긴다. 본 연구에서는 아카네에서 일어나는 측화의 자연 탈리현상에 관여하는 유전자를 찾고, 이를 micro-tomato 에 형질전환하여 탈리 현상에 관여하는 지의 여부를 탐색하였다. 또한 sevin을 처리한 후지의 측화에서 일어나는 인공 탈리에 관여하는 유전자를 찾아 자연 탈리현상에 대한 정보를 얻고자 하였다. 이를 위해 아카네 측화와 sevin 처리구를 tester로, 후지 측화와 sevin 무처리구를 driver 로 사용하여 suppression subtractive hybridization (SSH)을 수행하였다. PCR을 통해 tester에서 특이적으로 발현되는 유전자가 증폭이 되고 이들을 T/A cloning vector 에 삽입하여 plasmid library를 제작하였다. 이 library를 dot blotting을 통해 tester에서 실제 특이적이 발현을 보이는 clone을 찾아내었다. 아카네에서는 putative ser/thr kinase (ASK), phosphate transporter (APT), chitinase, cellulase, ribonuclease PR-10 like protein, unknown genes 이 up-regulation 되었고, cytochrome P-450 monooxygenase, hemolysin, sorbitol dehydrogenase, glycerol kinase (AGK), unknown genes 이 down-regulation 되었다. sevin 처리구에서는 putative senescence-associated protein, ribosomal protein L19, chloroplast ribosomal protein, unknown genes 이 up-regulation 되었고, elongation factor 1-α, fructose 1,6-bisphosphate aldolase, RING zinc finger protein, unknown genes 이 down-regulation 되었다. 아카네와 sevin 처리구에서 up- 또는 down-regulation 되는 유전자들을 비교한 결과 서로 일치되어 발현되는 것이 없어 자연탈리와 적과제에 의한 인공탈리는 서로 다른 메카니즘에 의해 일어나는 것으로 생각된다. 실제로 sevin 은 세포를 죽이는 데 관여한다는 보고가 있어 자연적으로 나타나는 탈리현상과는 다르게 탈리를 일으킬 것으로 생각된다. 이들 유전자중 putative ser/thr kinase (ASK), phosphate transporter (APT), glycerol kinase (AGK)를 선택하여 발현 패턴을 분석하고 유전자를 cloning 하였다. 아카네에서 up-regulation 되었던 chitinase, cellulase, ribonuclease PR-10 like protein 은 여러 논문에서 탈리 현상에 관여되있는 것으로 보고되어 있다. cDNA library 와 genomic DNA library 를 screening 하여 각각의 full gene을 얻었다. 이들 유전자를 pBI121 vector 에 삽입하여 Agrobacterium tumfaciens 을 형질전환하였고, 이를 이용해 micro-tomato를 형질전환 하였다. 형질전환을 통해 2개의 ASK 형질전환체와 3 냅?APT 형질전환체를 얻었다. ASK 형질전환체는 순화과정중 2개의 꽃이 자연탈리되는 현상을 보였고, wild type 과 비교했을 때 키가 작거나 잎이 무성한 phenotype을 보였다. APT 형질전환체는 외형적으로 다른 phenotype 은 보이지 않는다.
